Somatic Profiling Research Articles

Somatic Profiling Prospectively Guides Germline Genetic Testing in Patients with Mutations in High-Risk Cancer Predisposition Genes

Somatic Profiling Prospectively Guides Germline Genetic Testing in Patients with Mutations in High-Risk Cancer Predisposition Genes

Frequency of Common and Uncommon BRAF Alterations among Colorectal and Non-Colorectal Gastrointestinal Malignancies.

The predictive and prognostic role of BRAF alterations has been evaluated in colorectal cancer (CRC); however, BRAF alterations have not been fully characterized in non-CRC gastrointestinal (GI) malignancies. In the present study, we report the frequency and spectrum of BRAF alterations among patients with non-CRC GI malignancies. Patients with CRC and non-CRC GI malignancies who underwent somatic tumor profiling via a tissue-based or liquid-based assay were included in this study. Gain-of-function BRAF alterations were defined as pathogenic/likely pathogenic somatic short variants (SVs), copy number amplifications ≥8, or fusions (RNA or DNA). Among 51,560 patients with somatic profiling, 40% had CRC and 60% had non-CRC GI malignancies. BRAF GOF alterations were seen more frequently in CRC (8.9%) compared to non-CRC GI malignancies (2.2%) (p < 0.001). Non-CRC GI malignancies with the highest prevalence of BRAF GOF alterations were bile duct cancers (4.1%) and small intestine cancers (4.0%). Among BRAF GOF alterations, class II (28% vs. 6.8%, p < 0.001) and class III (23% vs. 14%, p < 0.001) were more common in non-CRC GI malignancies. Among class II alterations, rates of BRAF amplifications (3.1% vs. 0.3%, p < 0.001) and BRAF fusions (12% vs. 2.2%, p < 0.001) were higher in non-CRC GI malignancies compared to CRC. Non-CRC GI malignancies demonstrate a distinct BRAF alteration profile compared to CRC, with a higher frequency of class II and III mutations, and more specifically, a higher incidence of BRAF fusions. Future studies should evaluate clinical implications for the management of non-CRC GI patients with BRAF alterations, especially BRAF fusions.

CDH1 Genotype Exploration in Women With Hereditary Lobular Breast Cancer Phenotype

Pathogenic or likely pathogenic (P/LP) germline CDH1 variants are associated with risk for diffuse gastric cancer and lobular breast cancer (LBC) in the so-called hereditary diffuse gastric cancer (HDGC) syndrome. However, in some circumstances, LBC can be the first manifestation of this syndrome in the absence of diffuse gastric cancer manifestation. To evaluate the frequency of germline CDH1 variants in women with the hereditary LBC (HLBC) phenotype, somatic CDH1 gene inactivation in germline CDH1 variant carriers' tumor samples, and the association of genetic profiles with clinical-pathological data and survival. This single-center, longitudinal, prospective cohort study was conducted from January 1, 1997, to December 31, 2021, with follow-up until January 31, 2023. Women with LBC seen at the European Institute of Oncology were included. Testing for germline CDH1, BRCA1, and BRCA2 genes was performed. Somatic profiling was assessed for germline CDH1 carriers. Accurate estimates of prevalence of germline CDH1 variants among patients with HLBC and the association of somatic sequence alteration with HLBC syndrome. The Kaplan-Meier method and a multivariable Cox proportional hazards regression model were applied for overall and disease-free survival analysis. Of 5429 cases of primary LBC, familial LBC phenotype accounted for 1867 (34.4%). A total of 394 women with LBC were tested, among whom 15 germline CDH1 variants in 15 unrelated families were identified. Among these variants, 6 (40.0%) were P/LP, with an overall frequency of 1.5% (6 of 394). Of the 6 probands with P/LP CDH1 LBC, 5 (83.3%) had a positive family history of BC and only 1 (16.7%) had sporadic juvenile early-onset LBC. No germline BRCA1 and BRCA2 variants were identified in CDH1 carriers. An inactivating CDH1 mechanism (second hit) was identified in 4 of 6 explored matched tumor samples (66.7%) in P/LP germline carriers. The P/LP CDH1 LBC variant carriers had a significantly lower age at diagnosis compared with the group carrying CDH1 variants of unknown significance or likely benign (42.5 [IQR, 38.3-43.0] vs 51.0 [IQR, 45.0-53.0] years; P = .03). In this cohort study, P/LP germline CDH1 variants were identified in individuals not fulfilling the classic clinical criteria for HDGC screening, suggesting that identification of these variants may provide a novel method to test women with LBC with early age at diagnosis and/or positive family history of BC.

Mainstream Model of Genetic Testing for Prostate Cancer at a Large Tertiary Cancer Centre

BackgroundAn estimated 20% to 30% of men with advanced prostate cancer carry a mutation in DNA damage repair genes, of which half are estimated to be germline. Eligibility criteria for germline genetic testing expanded significantly for Ontario patients in May 2021 and many centers adopted a “mainstream” model, defined as oncologist-initiated genetic testing. MethodsWe conducted a retrospective chart review to report on the first-year mainstream experience of a large tertiary oncologic center, the Sunnybrook Odette Cancer Centre. All patients who underwent mainstream at the discretion of their treating physician were included. A subset underwent somatic profiling as part of clinical trial screening. Descriptive statistics were used to report baseline clinicopathologic characteristics and treatments received. ResultsBetween May 1, 2021, and May 30, 2022, 174 patients with prostate cancer underwent mainstream germline genetic testing with a 19-gene panel. Median age was 75 (IQR 68-80), and 82% of patients were diagnosed with either de novo metastatic or high-risk localized prostate adenocarcinoma. Fourteen patients (8%; 95% CI 4%-12%) were found to have a deleterious germline mutation, including pathogenic or likely pathogenic variants in BRCA1/2, ATM, CHEK2, PMS2, RAD51C, HOXB13, and BRIP1. Forty-nine patients (28%; 95% CI 21%-35%) were found to have a variant of uncertain significance. Thirty-four patients also had next-generation sequencing (NGS) of their somatic tissue. Among this subset, 8 of 34 (23%) had an alteration in homologous recombination repair (HRR) genes. Of the 14 patients with a germline mutation, none had a prior personal history of malignancy and 6 (43%) did not have any first- or second-degree relatives with history of prostate, pancreatic, breast, or ovarian cancer. ConclusionWe report on the real-world characteristics of prostate cancer patients who underwent mainstream germline genetic testing. Personal history and family history of cancer cannot reliably stratify patients for the presence of pathogenic germline variants.

Confirmed Pathogenic Germline Variants in Cancer Predisposition Genes Incidentally Detected in Somatic Genomic Profiling of Multiple Myeloma Patients

Introduction: We previously showed that ~10% of patients with multiple myeloma (MM) carry pathogenic or likely-pathogenic (P/LP) germline variants (PGVs) in known cancer predisposition genes, and that moderate/high-penetrance PGVs in DNA repair genes may be associated with MM risk and favorable response to alkylating chemotherapy. Increasingly applied in MM, genomic profiling by next-generation sequencing (NGS) can identify prognostically-relevant and therapeutically-actionable somatic mutations, but it may also incidentally unveil PGVs, which can have significant implications for patients and their families. We here test the diagnostic accuracy of a novel referral tool designed to flag likely PGVs warranting confirmatory germline testing among mutations identified in somatic NGS studies of 1161 MM patients from our institution. Methods: We retrospectively reviewed 1715 commercial NGS reports of 1161 MM patients treated at the Icahn School of Medicine at Mount Sinai (ISMMS) between 2015 &amp; 2022 (targeted 448-gene somatic profiling panels performed by a single genomic testing company for clinical purposes on bone marrow aspirate samples). In collaboration with certified genetic counselors at our institution, we applied evidence-based criteria to develop a tool to flag P/LP variants of likely germline origin warranting a referral for confirmatory germline testing (“Likely PGVs”). The resulting referral tool flags variants detected in 35 well-established cancer predisposition genes at a variant allele frequency (VAF) of &amp;gt;10%, as well as 24 cancer predisposition founder variants at any VAF. Next, we tested the positive predictive value (PPV), negative predictive value (NPV), sensitivity and specificity of our approach for the detection of true PGVs by analyzing paired germline whole-exome sequencing (WES) in 382/1161 (33%) MM patients in the study cohort. Germline WES data were generated for research purposes using a clinically-validated pipeline for PGV detection and annotation. Results: 385 P/LP variants were reported in somatic NGS studies of 382 MM patients who had paired somatic/germline sequencing data. Of these, 38/385 (9.9%) were found to be true PGVs in confirmatory germline testing. True PGVs detected in more than one gene included nine APC, seven CHEK2, five BRCA1, three BRCA2, three ATM and three MUTYH variants. The referral tool flagged 41/385 (10.6%) of the cohort's somatic variants as likely PGVs, identifying 32/38 (84.2%) true PGVs. The six PGVs missed by the referral tool were detected in the following genes: one APC, one CHEK2, one MITF, one MUTYH, one NF1 and one SHDA variant. In contrast, the nine variants that were mislabeled as likely PGVs but were in fact somatic were: two ATM, two BRCA2, two TSC1, one CHEK2, one BRIP1, and one RET. With a variant allele threshold of &amp;gt;10%, the tool had a PPV of 78.0% and a NPV of 98.3% for the detection of true PGVs. The sensitivity and specificity were 84.2% and 97.5%, respectively. Among the 692/1161 MM patients who did not have confirmatory germline testing (67%), the proportion of variants flagged as likely PGVs was very similar to that of the paired somatic/germline group (77/696, 11.1%), and variants were detected in similar genes. Assuming a PPV of 78%, 60 of these variants could represent true PGVs. Conclusion: A novel tool designed to flag likely PGVs among mutations identified in somatic genomic profiling of MM patients had high sensitivity and specificity for the detection of true PGVs. 9.9% of all MM patients with paired somatic/germline testing were found to have a true PGV, validating the results of our prior study and once again highlighting the need to implement screening strategies in high-risk subgroups of MM patients, such as those with strong family history of cancer or prior personal history of cancer.

Somatic profiling guides germline genetic testing in patients with mutations in cancer predisposition genes.

10608 Background: 15% of patients with advanced cancer have an inherited predisposition to cancer. Over a third of patients carrying a pathogenic germline variant would not qualify for genetic testing based on current National Comprehensive Cancer Network genetic testing guidelines. Supplementing the current practice of somatic sequencing with germline testing would improve treatment decisions and be informative of the risk of developing subsequent cancers in the individual. It may also extend the benefits of high-risk screening programs to family members. Offering genetic testing to patients with the highest likelihood of carrying a germline mutation is preferable to unselected sequencing given the current environment. Methods: We developed a list of genes to help identify patients whose somatic mutations were more likely to be found in the germline as well. We classified these genes into 3 tiers based on the likelihood of germline pathogenicity. All somatic sequencing panel results were reviewed based on the IRB-approved protocol for our designated Tier One genes ( ATM, BAP1, BMPR1A, BRCA1, BRCA2, BRIP1, MSH2, MSH6, MUTYH, MYH11, PALB2, RUNX1, SDHAF2, SDHB, SDHC, and known founder mutations in additional genes). Treating physicians for patients whose tumors harbored a mutation in one of these genes were contacted and, if they agreed, patients were offered participation in this ongoing study. Enrolled patients had genetic counseling and testing for a multi-gene panel at no cost to them. Results: We reviewed 3621 somatic test results, and 339 (9.4%) patients were eligible for the study. 34 (10.03%) eligible patients were enrolled, and 33 (9.73%) completed testing. Of these, 16 had positive germline results (48.5%, though they may not all be in the originally identified genes), 8 had a variant of uncertain significance (24.2%), and 9 had negative testing (27.3%). Some patients were not referred by their treating physicians based on their own clinical assessment. Others were hesitant to enroll due to concerns about the privacy of genetic information. Other confounding factors may have affected our accrual, including the pandemic. Conclusions: Implementing a somatic profiling-based screening program to identify inherited cancer predisposition syndromes is feasible in patients who may not meet current germline genetic testing guidelines. Our study showed 9.73% of eligible patients underwent genetic counseling and testing, and an inherited predisposition to cancer was confirmed in almost 50% of these patients. In addition, family members were offered cascade testing. Germline genetic evaluation remains underutilized even when payor constraints are removed as a barrier. To increase the identification of patients with inherited predispositions to cancer other barriers such as a lack of referrals by treating oncologists need to be overcome.

Abstract 133: Rewinding the tape of life: somatic profiling in Von Hippel-Lindau (VHL) disease reveals evolutionary contingency and constraints

Abstract Whether cancer evolution follows a deterministic route prescribed by host factors (germline genetics and/or environment) or proceeds along multiple alternative trajectories due to the stochastic nature of evolution is unknown. Patients with VHL disease have a pathogenic germline mutation in VHL (3p25) and tumorigenesis occurs when the wild-type copy of VHL is lost. Patients develop multiple clonally independent tumors including clear cell renal cell carcinoma (ccRCC), pheochromocytoma or paraganglioma (PPGL) and pancreatic neuroendocrine tumors (pNET), offering an opportunity to test repeatability of evolutionary trajectories between and within patients. We performed deep targeted exome sequencing, in 1520 tumors (993 solid ccRCC; 156 cystic ccRCC; 135 mixed solid/cystic ccRCC; 236 extra-renal) from 130 patients with VHL disease to evaluate somatic variants and copy number alterations, ploidy and chromosomal complexity. Tumor phenotype was evaluated from pathology reports and longitudinal growth kinetics from diagnostic MRI data. Analysis of 889 tumor samples is complete and profiling of the full cohort will be presented at the 2023 AACR meeting. Within clonally independent tumors, we performed two tests to assess for evidence of convergence between tumors from the same patient. First, we tested for parallel events in canonical ccRCC drivers. We found significant enrichment of somatic VHL variants occurring in one patient with a germline whole VHL gene deletion encompassing neighboring BRK1. Contiguous BRK1 loss is associated with less aggressive ccRCC phenotype and our findings suggest a specific constraint in this patient. We next assessed for evidence of convergence by measuring the genetic distance of copy number and mutational profiles between and within patients with resampling and permutation. Similar somatic profiles between tumors were only detected in 1/76 patients whose tumors were characterized by infrequent somatic alterations beyond 3p loss indicating little evidence of convergence. Cystic ccRCCs represent an indolent phenotype not associated with metastasis that may progress to solid tumors over time; we found mixed tumors closely resemble somatic profiles of solid tumors whereas cysts were characterized by infrequent copy number gains and low chromosomal complexity. We also identified cysts with specific losses (chr16, chr17, chr19p) mutually exclusive with 3p LOH suggestive of VHL-loss independent cyst formation. In solid ccRCCs, we found chromosomal complexity and loss of specific chromosomal loci (9p and/or 14q) are associated with diameter potentially reconciling the observation linking risk of metastasis to tumor diameter in VHL-related ccRCC. In summary, within the constrained setting of VHL-related ccRCC, evolution is dominated by chance although specific patient and phenotype specific constraints are observed. Citation Format: Scott T. Shepherd, Alex Coulton, Cathy D. Vocke, Chris J. Ricketts, Mark Ball, Fiona Byrne, Yuliia Dovga, Annika Fendler, Barber Taja, Olga O'Neill, Lina Gerontogianni, Gavin Kelly, Laura S. Schmidt, Daniel R. Crooks, Kirstin Choo, Rabindra Gautam, Zoe Blake, Krista Reynolds, Kevin Litchfield, Maria Merino, Marston W. Linehan, Samra Turajlic. Rewinding the tape of life: somatic profiling in Von Hippel-Lindau (VHL) disease reveals evolutionary contingency and constraints [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 133.

1683P Implementing next generation sequencing (NGS)/molecular tumor board (MTB)-based precision oncology practice: One-year experience at the Verona University Hospital

Cancer is a multifactorial genetic disease: NGS-based broad genomic profiling allows the identification of potential molecular drivers. Evaluation of the clinical actionability of NGS results should be guided by the expert consensus of a MTB, providing management recommendations and translating molecular alterations into clinical indications. Here we describe the MTB experience at the Verona University Hospital. Between Feb 2021 and Mar 2022, 315 patients (pts) with advanced solid malignancies deemed orphan with respect to established molecular diagnostics and availability of standard molecularly targeted treatments, had access to somatic profiling using targeted NGS panels. NGS results were discussed at bi-weekly MTB meetings, encompassing oncologists, pathologists, geneticists, pharmacologists, and pharmacists. The three most represented primary tumors were pancreatic (30%), breast (16%) and head and neck (14%) cancers. Among 315 pts profiled (13% of the annual active oncological population at our Center), 42 were discussed at MTB meetings, so far: 15 patients (36%) received a treatment recommendation: 4 within an ongoing clinical trial, 11 through atypical drug access (authorized off label use, named patient programs and other access schemes). Six pts (14%) were referred for genetic counseling, based on evidence of potential germline alterations at somatic testing. Further molecular investigations were requested/proposed in 11 pts (26%): IHC in 5, germline testing in 5, and a second NGS test + IHC in 1 pt, respectively. NGS-based profiling is a comprehensive tool to identify potentially actionable targets in advanced solid tumors. Its systematic application may have a profound impact on individual pts clinical course and outcomes. MTB discussion successfully provided suitable management strategies in a significant fraction of pts. Based on the careful definition of the criteria for accessing NGS profiling and the consequent MTB discussion, such approach should become a constitutive part of the oncological care program in the era of precision medicine.

Abstract 5108: Copy number analysis of pure DCIS and association with recurrence

Abstract With the widespread adoption of breast cancer screening the incidence of pure ductal carcinoma in situ (DCIS) has increased. As DCIS is considered a non-obligate precursor of invasive ductal carcinoma most women with pure DCIS are treated with breast conserving surgery (BCS) +/- radiotherapy. However, for many this is likely to be overtreatment as only a minority will develop a subsequent ipsilateral recurrence. Studies also show that only ~60% of these ipsilateral recurrences are invasive disease with the remainder being pure DCIS. To predict which women are most likely to benefit from interventions, there is a need to identify biomarkers that are associated with invasive recurrence. Our aim was to assess whether copy number aberrations (CNAs) could be used to identify DCIS that was likely to recur as invasive disease or remain recurrence-free during long-time follow up. We performed somatic copy number profiling on 309 pure DCIS samples that had not developed an ipsilateral event (controls), 198 that had developed subsequent ipsilateral invasive disease (INV-cases) and 58 that had developed subsequent ipsilateral pure DCIS (DCIS-cases). The samples were obtained from two large nation-wide cohorts: the Sloane cohort, a prospective breast screening cohort from the UK with a median follow up of 12.5 years and a Dutch population based cohort, with a median follow up of 13 years. CNAs were assessed using the CytoSNP array or low pass whole genome sequencing and analyzed using GISTIC. Integrative cluster (IntClust) subtyping revealed that only 5 subtypes were well represented in DCIS compared to 10 in invasive disease and the distribution of clusters between INV-cases and controls was similar with the exception of IntClust 4, which was significantly more common in controls (P= 0.025, Fishers exact test). IntClust 4 is characterized to have low levels of genomic instability and a CNA-devoid. INV-cases were globally more aberrant than controls (P = 0.006, Wilcoxon test) as assessed by the chromosomal instability index (CIN) score. GISTIC identified 17 recurrent amplifications, 21 recurrent gains and 22 recurrent losses in the whole cohort. Six of these regions were more common in INV-cases compared to controls: amplifications at 17q24.1 and 8p11.23, losses at 1p36.13 and 11q23.2 ​and gains at 17q21.33​ and 16p (Nominal P &amp;lt; 0.05 and FDR &amp;lt; 0.1, Fishers exact test). Subgroup analysis of ER+, Her2- INV-cases versus controls revealed an additional differential CNA, amplification at 11q13.3 more common in cases. DCIS-cases had similar CNAs to INV-cases and were more aberrant than controls in terms of CIN score (P &amp;lt; 0.037, Wilcoxon test) but not as aberrant as INV-cases. In conclusion, we have identified potential CNAs that are associated with invasive recurrence. Further analysis will integrate gene expression with copy number data to identify which genes are being targeted by these CNAs in order to identify pathways important in progression of DCIS. Citation Format: Ahmed A. Ahmed, Maria Roman-Escorza, Tycho Bismeijer, Michael Sheinman, Vandna Shah, Rana Shami, Jeffrey R. Marks, Lorraine M. King, Anargyros Megalios, Lindy L. Visser, Marlous Hoogstraat, Helen R. Davies, Tapsi Kumar, Deborah Collyar, Hilary Stobart, Sarah Pinder, Nicholas N. Navin, Andrew Futreal, Serena Nik-Zainal, E. Shelley Hwang, Lodewyk F. Wessels, Esther H. Lips, Alastair Thompson, Jelle Wesseling, Elinor J. Sawyer. Copy number analysis of pure DCIS and association with recurrence [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5108.

An optimized protocol for evaluating pathogenicity of VHL germline variants in patients suspected with von Hippel-Lindau syndrome: Using somatic genome to inform the role of germline variants

The interpretation of hereditary genetic sequencing variants is often limited due to the absence of functional data and other key evidence to assess the role of variants in disease. Cancer genetics is unique, as two sets of genomic information are often available from a cancer patient: somatic and germline. Despite the progress made in the integrated analysis of somatic and germline findings, the assessment of pathogenicity of germline variants in high penetrance genes remains grossly underutilized. Indeed, standard ACMG/AMP guidelines for interpreting germline sequence variants do not address the evidence derived from tumor data in cancer. Previously, we have demonstrated the utility of somatic tumor data as supporting evidence to elucidate the role of germline variants in patients suspected with VHL syndrome and other cancers. We have leveraged the key elements of cancer genetics in these cases: genes with expected high disease penetrance and those with a known biallelic mechanism of tumorigenicity. Here we provide our optimized protocol for evaluating the pathogenicity of germline VHL variants using informative somatic profiling data. This protocol provides details of case selection, assessment of personal and family evidence, somatic tumor profiles, and loss of heterozygosity (LOH) as supporting evidence for the re-evaluation of germline variants.

Characterization of Genetic Heterogeneity in Recurrent Metastases of Renal Cell Carcinoma.

Simple SummarySurvival rates in metastatic renal cell carcinoma (RCC) are still low despite novel therapies available. Thus, knowledge of molecular characteristics of distant metastases is important for personalized treatment strategies. Therefore, we investigated the genetic landscape of metastases, including synchronous and/or recurrent metastases to elucidate potential drug target genes and clinically relevant mutations. Furthermore, differences in mutational composition in different metastatic sites and over the course of the disease and treatment will demonstrate the importance of somatic profiling for precision medicine in RCC, thereby improving disease management in the future.Metastatic renal cell carcinoma (RCC) exhibits poor prognosis. Better knowledge of distant metastases is crucial to foster personalized treatment strategies. Here, we aimed to investigate the genetic landscape of metastases, including synchronous and/or recurrent metastases to elucidate potential drug target genes and clinically relevant mutations in a real-world setting of patients. We assessed 81 metastases from 56 RCC patients, including synchronous and/or recurrent metastases of 19 patients. Samples were analysed through next-generation sequencing with a high coverage (~1000× mean coverage). We therefore established a novel sequencing panel comprising 32 genes with impact on RCC development. We observed a high frequency of mutations in known RCC driver genes (e.g., >40% carriers of VHL and PBRM1 mutations) in metastases irrespective of the metastatic site. The somatic mutational composition was significantly associated with cancer-specific survival (p(logrank) = 0.03). Moreover, we identified in 34 patients at least one drug target gene as well as clinically relevant mutations listed in the VICC Meta-Knowledgebase in 7%. In addition to significantly higher mutational burden in recurrent metastases compared to earlier ones, synchronous and/or recurrent metastases of individual patients, even after a time-period >2 yrs, shared a high proportion of somatic events. Our data demonstrate the importance of somatic profiling in metastases for precision medicine in RCC.

Interplay between copy number alterations and immune profiles in the early breast cancer Scandinavian Breast Group 2004-1 randomized phase II trial: results from a feasibility study

Emerging data indicate that genomic alterations can shape immune cell composition in early breast cancer. However, there is a need for complementary imaging and sequencing methods for the quantitative assessment of combined somatic copy number alteration (SCNA) and immune profiling in pathological samples. Here, we tested the feasibility of three approaches—CUTseq, for high-throughput low-input SCNA profiling, multiplexed fluorescent immunohistochemistry (mfIHC) and digital-image analysis (DIA) for quantitative immuno-profiling- in archival formalin-fixed paraffin-embedded (FFPE) tissue samples from patients enrolled in the randomized SBG-2004-1 phase II trial. CUTseq was able to reproducibly identify amplification and deletion events with a resolution of 100 kb using only 6 ng of DNA extracted from FFPE tissue and pooling together 77 samples into the same sequencing library. In the same samples, mfIHC revealed that CD4 + T-cells and CD68 + macrophages were the most abundant immune cells and they mostly expressed PD-L1 and PD-1. Combined analysis showed that the SCNA burden was inversely associated with lymphocytic infiltration. Our results set the basis for further applications of CUTseq, mfIHC and DIA to larger cohorts of early breast cancer patients.

Early-Onset Pancreas Cancer: Clinical Descriptors, Genomics, and Outcomes.

Recent evidence suggests a rising incidence of cancer in younger individuals. Herein, we report the epidemiologic, pathologic, and molecular characteristics of a patient cohort with early-onset pancreas cancer (EOPC). Institutional databases were queried for demographics, treatment history, genomic results, and outcomes. Overall survival from date of diagnosis was estimated using Kaplan-Meier method. Between 2008 and 2018, 450 patients with EOPC were identified at Memorial Sloan Kettering. Median overall survival was 16.3 (95% confidence interval [CI] = 14.6 to 17.7) months in the entire cohort and 11.3 (95% CI = 10.2 to 12.2) months for patients with stage IV disease at diagnosis. Of the patients, 132 (29.3% of the cohort) underwent somatic testing; 21 of 132 (15.9%) had RAS wild-type cancers with identification of several actionable alterations, including ETV6-NTRK3, TPR-NTRK1, SCLA5-NRG1, and ATP1B1-NRG1 fusions, IDH1 R132C mutation, and mismatch repair deficiency. A total of 138 patients (30.7% of the cohort) underwent germline testing; 44 of 138 (31.9%) had a pathogenic germline variant (PGV), and 27.5% harbored alterations in cancer susceptibility genes. Of patients seen between 2015 and 2018, 30 of 193 (15.5%) had a PGV. Among 138 who underwent germline testing, those with a PGV had a reduced all-cause mortality compared with patients without a PGV controlling for stage and year of diagnosis (hazard ratio = 0.42, 95% CI = 0.26 to 0.69). PGVs are present in a substantial minority of patients with EOPC. Actionable somatic alterations were identified frequently in EOPC, enriched in the RAS wild-type subgroup. These observations underpin the recent guidelines for universal germline testing and somatic profiling in pancreatic ductal adenocarcinoma.

Molecular medicine tumor board: whole-genome sequencing to inform on personalized medicine for a man with advanced prostate cancer

PurposeMolecular profiling of cancer is increasingly common as part of routine care in oncology, and germline and somatic profiling may provide insights and actionable targets for men with metastatic prostate cancer. However, all reported cases are of deidentified individuals without full medical and genomic data available in the public domain.Patient and methodsWe present a case of whole-genome tumor and germline sequencing in a patient with advanced prostate cancer, who has agreed to make his genomic and clinical data publicly available.ResultsWe describe an 84-year-old Caucasian male with a Gleason 10 oligometastastic hormone-sensitive prostate cancer. Whole-genome sequencing provided insights into his tumor’s underlying mutational processes and the development of an SPOP mutation. It also revealed an androgen-receptor dependency of his cancer which was reflected in his durable response to radiation and hormonal therapy. Potentially actionable genomic lesions in the tumor were identified through a personalized medicine approach for potential future therapy, but at the moment, he remains in remission, illustrating the hormonal sensitivity of his SPOP-driven prostate cancer. We also placed this patient in the context of a large prostate-cancer cohort from the PCAWG (Pan-cancer Analysis of Whole Genomes) group. In this comparison, the patient’s cancer appears typical in terms of the number and type of somatic mutations, but it has a somewhat larger contribution from the mutational process associated with aging.ConclusionWe combined the expertise of medical oncology and genomics approaches to develop a molecular tumor board to integrate the care and study of this patient, who continues to have an outstanding response to his combined modality treatment. This identifiable case potentially helps overcome barriers to clinical and genomic data sharing.

Genomic Features and Classification of Homologous Recombination Deficient Pancreatic Ductal Adenocarcinoma

Homologous recombination deficiency (HRD) in pancreatic ductal adenocarcinoma (PDAC), remains poorly defined beyond germline (g) alterations in BRCA1, BRCA2, and PALB2. We interrogated whole genome sequencing (WGS) data on 391 patients, including 49 carriers of pathogenic variants (PVs) in gBRCA and PALB2. HRD classifiers were applied to the dataset and included (1) the genomic instability score (GIS) used by Myriad's MyChoice HRD assay; (2) substitution base signature 3 (SBS3); (3) HRDetect; and (4) structural variant (SV) burden. Clinical outcomes and responses to chemotherapy were correlated with HRD status. Biallelic tumor inactivation of gBRCA or PALB2 was evident in 43 of 49 germline carriers identifying HRD-PDAC. HRDetect (score ≥0.7) predicted gBRCA1/PALB2 deficiency with highest sensitivity (98%) and specificity (100%). HRD genomic tumor classifiers suggested that 7% to 10% of PDACs that do not harbor gBRCA/PALB2 have features of HRD. Of the somatic HRDetecthi cases, 69% were attributed to alterations in BRCA1/2, PALB2, RAD51C/D, and XRCC2, and a tandem duplicator phenotype. TP53 loss was more common in BRCA1- compared with BRCA2-associated HRD-PDAC. HRD status was not prognostic in resected PDAC; however in advanced disease the GIS (P= .02), SBS3 (P= .03), and HRDetect score (P= .005) were predictive of platinum response and superior survival. PVs in gATM (n= 6) or gCHEK2 (n= 2) did not result in HRD-PDAC by any of the classifiers. In 4 patients, BRCA2 reversion mutations associated with platinum resistance. Germline and parallel somatic profiling of PDAC outperforms germline testing alone in identifying HRD-PDAC. An additional 7% to 10% of patients without gBRCA/PALB2 mutations may benefit from DNA damage response agents.

Abstract IA-08: Clinical advances in pancreas adenocarcinoma

Abstract IA-08: Clinical advances in pancreas adenocarcinoma

A Melanoma-Tailored Next-Generation Sequencing Panel Coupled with a Comprehensive Analysis to Improve Routine Melanoma Genotyping.

Tumor molecular deciphering is crucial in clinical management. Pan-cancer next-generation sequencing panels have moved towards exhaustive molecular characterization. However, because of treatment resistance and the growing emergence of pharmacological targets, tumor-specific customized panels are needed to guide therapeutic strategies. The objective of this study was to present such a customized next-generation sequencing panel in melanoma. Melanoma patients with somatic molecular profiling performed as part of routine care were included. High-throughput sequencing was performed with a melanoma tailored next-generation sequencing panel of 64 genes involved in molecular classification, prognosis, theranostic, and therapeutic resistance. Single nucleotide variants and copy number variations were screened, and a comprehensive molecular analysis identified clinically relevant alterations. Four hundred and twenty-one melanoma cases were analyzed (before any treatment initiation for 94.8% of patients). After bioinformatic prioritization, we uncovered 561 single nucleotide variants, 164 copy number variations, and four splice-site mutations. At least one alteration was detected in 368 (87.4%) lesions, with BRAF, NRAS, CDKN2A, CCND1, and MET as the most frequently altered genes. Among patients with BRAFV600 mutated melanoma, 44.5% (77 of 173) harbored at least one concurrent alteration driving potential resistance to mitogen-activated protein kinase inhibitors. In patients with RAS hotspot mutated lesions and in patients with neither BRAFV600 nor RAS hotspot mutations, alterations constituting potential pharmacological targets were found in 56.9% (66 of 116) and 47.7% (63 of 132) of cases, respectively. Our tailored next-generation sequencing assay coupled with a comprehensive analysis may improve therapeutic management in a significant number of patients with melanoma. Updating such a panel and implementing multi-omic approaches will further enhance patients' clinical management.

Abstract 2073: Exome sequencing reveals PTPRS, NOTCH3, FGFR1/3 mutations as novel mutational features in medullary thyroid cancer

Abstract Background: Approximately 1,000 patients are diagnosed with medullary thyroid cancer (MTC) in the United States each year; a tumor type that disproportionately causes thyroid cancer-related death. The molecular hallmark of MTC are mutations of the Rearranged During Transfection (RET) proto-oncogene: ~25% of MTC patients harbor germline RET variants and somatic RET mutations can be found in ~ 40% of sporadic medullary thyroid cancers. However, with the exception of RAS mutations relatively little is known of the broad molecular landscape of MTC. Material &amp; Methods: To further explore the mutational features of MTC, we performed whole exome sequencing on paired tumor and normal tissue on a cohort of 14 patients with MTC. For variant calling, we focused on the 468 genes included in the MSK-IMPACT panel. Paired RNA sequencing was available for 7 patients. A variant was included as potential pathogenic based on a combination of SIFT, Polyphen and clinical significance prediction. Only coding variants were considered. Results: Out of the 14 patients, 43% were female (n=6), and 57% (n=8) were male (Table 1). The median age was 55.5 years (range: 21-79 years). At time of diagnosis, 29% had stage I (n=4), 14% stage II (n=2), 21% stage III (n=3), and 36% (n=5) had stage IV disease. The majority of patients (71%, n=10) had multifocal disease. While half of the patients had lymphovascular tumor invasion (50%, n=7), only 14% (n=2) had capsular invasion. Of all patients, 29% (n=4) of patients were germline RET mutation carriers. In our somatic tumor profiling, we detected a total of 23 potential pathogenic variants. The median mutation burden per patient was 2 (range= 0-7). Three patients had no detectable mutations. Expectedly, mutations in the RET gene were also the most frequent somatic mutational events and were detected in 43% (n=6) patients. Interestingly, we identified recurrent mutations in 3 genes that to the best of our knowledge have not been previously described in MTC. Protein- Tyrosine phosphate receptor-type sigma (PTPRS), Notch receptor 3 (NOTCH3), and Fibroblast growth factor receptors1/3 (FGFR1/3). Each of these was mutated in 21% (n=2) MTC patients. In addition, 10 genes were found mutated in a single MTC patient (CHEK2, DNMT1, EP300, GNAS, KMT2A, PIK3C2G, PNRC1, POLE, PRKAR1A, SOX2). In the RET wildtype patients, DNMT1, FGFR1, KMT2A, PIK3C2G, PRKAR1A and PTPRS were the only detected lesions, suggesting a possible pathophysiological importance independent of RET. Conclusions: Our exome sequencing study identified recurrent mutations in PTPRS, NOTCH3, FGFR1/3 as novel recurrent mutational features in MTC. Notably, some of them were detected in RET wildtype patients, suggesting a possible functional role of the genes in carcinogenesis. The functional studies to further define the roles of these gene mutations in MTC are ongoing. Citation Format: Isaiah Boateng, Ayse S. Yilmaz, Pamela Brock, Sandya Liyanarachchi, Ravi Patel, Christopher J. Walker, John Phay, Albert de la Chapelle, Matthew Ringel, Ann-Kathrin Eisfeld. Exome sequencing reveals PTPRS, NOTCH3, FGFR1/3 mutations as novel mutational features in medullary thyroid cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2073.

Progression-free survival estimates in non-small cell lung cancer when RECIST is unavailable: Project GENIE’s integration of genomic, therapeutic and phenomic data.

9622 Background: Molecular tumor profiling has become an integral component of oncology practice but linked genomic-phenomic data remain scarce. Recurrence, treatment response and progression are not structured consistently in medical records and this deficit has been a roadblock to discovery of biomarkers that are associated with favorable outcomes. Methods: The Genomics Evidence Neoplasia Information Exchange (GENIE) consortium is an AACR sponsored project to link and share genomic and phenomic data to promote discovery in precision medicine. 3 cancer centers that routinely perform somatic tumor profiling for advanced cancers agreed to curate anti-neoplastic treatment exposures and outcomes including recurrence, progression, response and survival using a standard method. 6 cancer types (lung, colorectal, breast, prostate, pancreas and bladder) were selected and a REDCAP database captures anti-neoplastic treatments, and specific elements from pathology, radiology and oncology reports. Curators abstract data using data fields that rely on the PRISSMM standard. “Real world” progression free survival (PFS) was identified based on curation of: 1) the text of radiologists’ reports for CT, PET/CT, PET and MRI scans (PFSI) and 2) medical oncologists’ notes (PFSM). PFSI and PFSM were estimated from the start of 1st line anti-neoplastic systemic therapy until progression or death for all patients with molecularly characterized non-small cell lung cancer (NSCLC). Results: Genomic sequencing was performed between 2015 and 2017 for 748 patients with NSCLC treated at three major cancer centers. Median age at diagnosis was 66 years (interquartile range 58, 73) and 43% were male. As shown in the table, when RECIST assessments are unavailable, estimates of PFS vary based on whether they are derived from radiologists’ or oncologists’ interpretations. Conclusions: Radiologists’ reports and oncologists’ reports provide different PFS estimates. Cohort studies should specify the method used to define “real world” endpoints. Project GENIE will have 1800 NSCLC patients with curated endpoints by the ASCO meeting. [Table: see text]

Molecular profiling of metastatic breast cancer (MBC) and target-based therapeutic matching in an Asian tertiary phase I oncology unit.

3561 Background: Somatic profiling of MBC has highlighted actionable mutations and driven trials of matched targeted therapy (tx). Previous phase I studies have reported improved outcomes following matched therapies with tumour molecular profiles. Here, we review next generation sequencing (NGS) and treatment outcomes of Asian MBC patients (pts) in the phase I unit of a tertiary centre. Methods: Pts with MBC referred to a phase I unit underwent NGS (n = 152). Tumour tissue was sequenced via the amplicon based Ion Ampliseq Cancer (IAC) v2 (50 genes) platform from 2014-2017 prior to institutional change to Foundation Medicine 1 (FM1) (324 genes) 2017-2019. Patients were counselled on findings and enrolled onto matched therapeutic trials where available. Results: NGS was successfully performed in 107 pts (IAC 46%, FM1 54%) of which tumour subtypes include hormone receptor positive 63%, triple negative breast cancer (TNBC) 28% and Her2 positive 19%. Median lines of prior tx for MBC was 4 (range 0-12). 89% had prior chemotherapy (CT), 57% prior endocrine therapy (ET). 72/107 (67%) sequenced patients had further treatment and 18 (25%) were matched to tx based on NGS findings (15 clinical trial, 3 off trial). Matching rates on both NGS platforms were similar (IAC 22% vs FM1 28%). Mutated pathways with potential matched tx included PIK3CA/AKT/PTEN (52%), DNA damage response (DRR) (15%), and FGFR (11%) pathways. PIK3 mutations were seen in 43% and associated with higher number of metastatic sites (p = 0.03); most prevalent aberrations were PIK3CA H1047R (41%) and PIK3CA E542K (13%). Matched cases were more heavily pretreated (mean lines of prior tx 5.3 matched vs 3.7, unmatched p = 0.05), and showed a median progression free survival (mPFS) of 24 weeks [w] and clinical benefit rate (complete/partial response or stable disease ≥ 12 weeks) of 53% on matched tx. Comparison by NGS platform showed improved mPFS for matched vs unmatched pts sequenced on FM1 vs IAC (FM1: 26 vs 19w, HR = 0.76 [95% CI: 0.3-1.9]; IAC: 8 vs 12w; HR = 1.21 [95% CI: 0.5-2.8]). Interestingly, 1 pt with SMARCB mutation, reportedly associated with the FGFR pathway, had a PFS of 70w on tx with a pan-FGFR inhibitor after progressing on 3 prior lines of tx (ET and CT). Conclusions: Molecular profiling of MBC in a phase I unit led to matched tx in 25% of cases. Matched pts showed encouraging mPFS with a suggestion of benefit in those matched after sequencing on a broader gene panel (FM1).